The prevention and control of inflammation is often of great importance for the treatment of humans and animals. Much research has been devoted to development of compounds having anti-inflammatory properties. Certain methods and chemical compositions have been developed which aid in inhibiting or controlling inflammation, but additional anti-inflammatory methods and compositions are needed.
Neuroinflammatory conditions are complex and poorly understood disease processes which are hypothesized to involve microglia (BM"PHgr") (Mallat, M. [1994] J. Leukoc. Biol. 56:416-422). BM"PHgr" are mononuclear-phagocytes which become activated in a number of inflammatory conditions, such as neurogenic inflammation, meningitis, septic shock, Down""s syndrome, postischemic brain injury, HIV encephalopathy, Parkinson""s disease, Alzheimer""s disease, amyotrophic lateral sclerosis and multiple sclerosis (Mayer, A. M. [1988] Medicina. (B. Aires.) 58:377-385), acquiring a macrophage-like phenotype and become cytotoxic to brain cells by releasing mediators, i.e., proteolytic enzymes, reactive oxygen intermediates, eicosanoids and cytokines (Mayer et al. [1999] Shock 11(3):180-186). Present therapies for these and other inflammatory conditions are based on steroids and nonsteroidal anti-inflammatory compositions which are usually associated with a high incidence of unsatisfactory toxicity and poor efficacy (Faden, A. I. and S. Salzman [1992] Trend in Pharmacological Sciences 13:29-35).
It has been found that some natural products and organisms are potential sources for chemical molecules having useful biological activity of great diversity. Marine sponges have proved to be such a source, and a number of publications have issued disclosing organic compounds derived from marine sponges. Such publications include Scheuer, P. J. Ed. [1978-1983] Marine Natural Products, Chemical and Biological Perspectives, Academic Press, New York; Faulkner, D. [1995] J. Nat. Prod. Rep. 12:223-269; [1994] 11:355-394; [1993] 10:497-539; [1992] 9:323-364; [1991] 8:97-147; [1990] 7:269-309; [1988] 5:613-663; [1987] 4:539-576; [1986] 3:1-33; [1984] O2xe2x88x92 1:551-598.
It has been suggested (Glaser, K. B., R. S. Jacobs [1987] Biochem. Pharmacol. 36:2079-2086) that manoalide, and other marine natural products have the potential to modulate leukocyte eicosanoid (Mayer, A. M. S., S. Oh, K. H. Ramsey et al. [1999] Shock 11(3):180-186) and O2xe2x88x92 and thromboxane B2 (TXB2) in BM"PHgr".
Certain cyclic alkaloid compositions, e.g., manzamines A-F derived from extracts of the marine sponge Haliclona sp., have been found to possess useful antitumor activity. These compounds have been described in, for example, U.S. Pat. Nos. 4,895,854; 4,895,853; and 4,895,852. Manzamines A-F have the following structures: 
These compounds have not previously been reported to play any role in inflammatory processes.
The objects of the present invention are accomplished by the provision of anti-inflammatory manzamine compounds. Specifically exemplified herein are manzanines A-F. The subject invention further concerns the use of various derivatives and analogs of these compounds. Advantageously, these compounds have been found to possess anti-antigen driven, immune mediated inflammation and anti-neurogenic inflammation activity. Manzamine A has been found to be particularly effective because of its potent anti-inflammatory activity and low toxicity.
As described herein, the invention also comprises pharmaceutical compositions, e.g. anti-inflammatory compositions, containing as an active ingredient an effective amount of one or more compounds described herein and a non-toxic, pharmaceutically acceptable carrier or diluent. The pharmaceutical compositions of the subject invention can further comprise other active compounds. Such other active compounds include, but are not limited to, other anti-inflammatory compounds for example, steroidal compounds, including hydrocortisone and the like; or non-steroidal anti-inflammatories, including acetylsalicylic acid (aspirin), ibuprofen, acetaminophen, indomethacin, and the like. The second active ingredient can include antiviral, antibacterial, antifungal or other antimicrobial compounds or antitumor compounds as well.
As described herein, the invention further comprises processes for the production of compounds and compositions of the invention and novel methods of use thereof, e.g. methods of inhibiting an inflammatory response in a human or animal.
In accordance with the invention, methods for inhibiting inflammation comprise administering to a human or animal in need of such treatment an effective amount of the pharmaceutical compositions described herein.
The subject invention pertains to novel uses as anti-inflammatory agents of manzamine compounds and compositions comprising the manzamine compounds. Surprisingly, the manzamine compounds of the subject invention can be highly effective in inhibiting antigen driven, immune-mediated inflammation and neurogenic inflammation activity.
In one embodiment, the subject invention pertains to the use of compounds having the following General Structure (I): 
wherein X1, X2, X3, X4, X5, and X6 are, independently, a hydrogen, halogen, hydroxy, lower alkoxy, lower acyloxy, or lower mono or dialkyl amino group; R1 is hydrogen, lower alkyl, or lower acyl group; R2 is hydrogen, hydroxy, lower alkoxy, or lower acyloxy group.
In a preferred embodiment of the invention, the invention pertains to anti-inflammatory compositions comprising manzamine A having the following structure: 
Further embodiments of the subject invention pertain to the anti-inflammatory use of compounds having General Structures (II)-(IV): 
wherein X1, X2, X3, X4, X5, and X6 are the same or different and are a hydrogen, halogen, hydroxyl, lower alkoxy, lower acyloxy, thiol, lower alkylthiol, nitro, amino, lower alkylsulfonyl, aminosulfonyl, hydroxy sulfonyl (xe2x80x94SO3H), lower acylamino, lower alkyl, or lower monoalkyl- or dialkyl-amino group; R1 and R2 are the same or different and are a hydrogen, lower alkyl, or lower acyl group; and Y is a hydrogen, hydroxyl, lower alkoxy, or lower acyloxy group.
In more specific embodiments of the invention, the invention comprises the anti-inflammatory use of the compounds designated as manzamine B, C, or D of the formulae: 
Further embodiments of the subject invention utilize General Structure V: 
wherein R is a hydrogen, halogen, hydroxy, or lower acyloxy group; and X is a double bonded oxygen, or is the same or different and is any two of a hydrogen, hydroxy, lower alkyl, lower alkoxy, or lower acyloxy group wherein said lower alkyl, alkoxy, or acyloxy groups have preferably, from 1 to 5 carbon atoms.
In a specific embodiment, the subject invention concerns manzamines E and F which have the following structures: 
In other embodiments of the invention, the double bonds in the composition of General Structures (I)-(V) are partially or fully reduced. In further embodiments of the invention, the composition is a mineral acid (e.g., HCl, H2SO4, H3PO4, HNO3, etc.) or organic salt of compositions according to the General Structures.
Methods for obtaining these compounds are described in, for example, U.S. Pat. Nos. 4,895,852; 4,895,853; and 4,895,854, which are herein incoporated in their entirety by reference thereto.
Skilled chemists having the benefit of the instant disclosure, can readily use procedures to prepare the subject compounds. In carrying out such operations, suitable filtration, chromatographic and other purification techniques can be used. These techniques could include, for example, reversed phase (RPLC), column, vacuum flash, medium pressure (MPLC) and high performance liquid chromatography (HPLC) with a suitable column such as silica gel, Sephadex LH-20, ammonia-treated silica gel, bonded phase RP-18, RP-8 and amino columns. Such columns are eluted with suitable solvents such as heptane, ethyl acetate, methylene chloride, methanol, isopropanol, acetonitrile water, trifluoroacetic acid (TFA) and various combinations thereof.
A novel use for the described compounds and compositions is their administration to an animal or human as an agent in the control of an inflammatory response. The discovery that the subject compounds have inhibitory activity against immune-mediated inflammation and neurogenic inflammation is particularly unexpected and advantageous. Specifically, neurogenic inflammation can be evoked by neuropeptides, such as substance P (SP), calcitonin gene-related peptide (CGRP), vasoactive intestinal peptide (VIP), and neurokinin A (NKA), released from primary afferent C-fiber nerve terminals and histamine, secondarily released from mast cells (Dray, A., [1992] xe2x80x9cNeuro pharmacological mechanisms of capsaicin and related substancesxe2x80x9d Biochem Pharm 44(4):611-15). In addition, it is known that capsaicin (CAP), the active constituent found in cayenne pepper, induces an acute neurogenic inflammatory response when applied topically to skin. CAP is a highly selective pain producing substance that selectively stimulates nociceptive and thermal-sensitive nerve endings in tissues by acting on a specific membrane receptor. The mode of action of capsaicin, therefore, differs significantly from phorbol myristate acetate (PMA)-induced immune-inflammation. By comparison, PMA elicits its pro-inflammatory effects through cellular activation of specific immune cells, such as macrophages and microglia. Consequently, the pain response to PMA develops more slowly than the immediate, but transient, pain response to capsaicin.
For purposes of the subject invention, unless otherwise noted, the terms xe2x80x9cinflammationxe2x80x9d and xe2x80x9cinflammatory responsexe2x80x9d include any and all reactions including, but not limited to, immune-related responses and/or allergic reactions to a physical, chemical, or biological stimulus. xe2x80x9cAnti-immune-mediatedxe2x80x9d and anti-neurogenic inflammatory activity,xe2x80x9d as used herein, will be understood by those of ordinary skill in the art to mean biological activity inhibiting or controlling an immune-mediated and/or neurogenic inflammatory response. Inflammation for which the primary activating inflammation is antigen-derived can be due to, for example, bacterial lipopolysaccharide.
Anti-inflammatory activity can occur by modes of action which can include, but are not limited to, lipid-mediated inflammatory responses, e.g., (i) suppression of cellular activation of phospholipase A2, either directly (as is known for the anti-inflammatory compound, manoalide) or indirectly (as is known for the anti-inflammatory compound, hydrocortisone); (ii) by inhibiting, or controlling, cyclooxygenation of arachidonic acid, similar to the action of non-steroidal anti-inflammatory drugs; or (iii) by affecting lipooxygenase products of peroxidase reactions to arachidonic acid, or by non-lipid-mediated inflammatory responses, which include inflammatory mediators such as cytokines (e.g. tumor necrosis factor alpha, interleukin 1), reactive oxygen species (e.g. superoxide anion (O2xe2x88x92), hydrogen peroxide (H2O2) and nitric oxide (NO), proteases, growth factors, complement and excitatory amino acids.
The compounds and compositions of the subject invention can be used in the treatment of inflammation at sites where the primary activating factor is antigen-derived (e.g. bacterial lipopolysaccharide) or of neurogenic origin. In a particularly preferred embodiment, the compounds of the subject invention are used to treat pathological inflammatory conditions of the brain. In a specific embodiment, these pathological inflammatory conditions involve microglia (BM"PHgr"). Thus, the compounds of the subject invention can be used to treat conditions including, but not limited to, neurogenic inflammation, meningitis, septic shock, Down""s syndrome, postischemic brain injury, HIV encephalopathy, Parkinson""s disease, Alzheimer""s disease, amyotrophic lateral sclerosis and multiple sclerosis.
The subject compounds and compositions can also be useful in the treatment of chronic pain, migraines, thermal-induced pain, such as sunburn, or other thermal and nociceptive pain, and chronic pain associated with arthritis. Uses can also include other inflammatory conditions that involve a neurogenic pain-producing component, e.g., certain metastatic carcinomas or inflammation of the blood vessels.
The compounds of the subject invention can also be used to treat a variety of skin conditions including, but not limited to, radiation irritation and burns (including UV and ionizing), chemical burns, rhinitis, thermal burns, reddening of the skin, and chemically induced lesions.
The compounds of the subject invention can also be used to treat allergic responses and/or promote wound healing. This can include the use of the compounds in aerosol form for the treatment of acute allergic reactions such as acute asthmatic attack and in the treatment of inflammation of the lung caused by chemical exposure.
The compounds of the subject invention can also be used to treat systemic anaphylactic reactions in animals and man.
The compounds of the subject invention can also be used to treat conjunctivitis, inflammatory gum diseases, inflammatory bowel disease, and nephritis.
The compounds of the subject invention can also be used to treat conditions where brain microglia are involved such as neurogenic inflammation, meningitis, septic shock, Down""s syndrome, post ischemic brain injury, HIV encephalopathy, Parkinson""s disease, Alzheimer""s disease, amyotrophic lateral sclerosis, and multiple sclerosis.
Chemicals. LPS B (E. coli. 026:B6) from Difco Lab, Detroit, Mich.; cytochrome C type III (from horse heart), superoxide dismutase (from bovine liver), phorbol 12-myristate 13-acetate (PMA) and dimethyl sulfoxide (DMSO) from Sigma Chemical Co., St. Louis, Mo. A stock solution of PMA (10 mM) in DMSO was maintained at xe2x88x9280xc2x0 C. and diluted prior to use. Dulbecco""s modified Eagle medium (DMEM) with high glucose (4.5 mg/l), Hanks"" balanced salt solution (HBSS), penicillin (P), streptomycin (S), trypsin (0.25%)-EDTA (1 mM) and trypan blue from GIBCO Laboratories, Life Technologies, Inc., Grand Island, N.Y.; heat-inactivated fetal bovine serum certified (FBS) from Hyclone, Logan, Utah. Stock solutions of Manzamine A, B, C, D, E, and F (10 mM) in DMSO were stored at xe2x88x9280xc2x0 C. and diluted prior to each experiment.
Isolation and culture of rat BM"PHgr". Briefly to prepare primary BM"PHgr" cultures, cerebral cortices from 1-2-day-old Sprague-Dawley rats (Harlan, Indianapolis, Ind.) were placed in cold DMEM+10% heat-inactivated FBS+120 U/ml P and 12 xcexcg/ml S, meninges carefully removed, brain tissue minced and dissociated with trypsin-EDTA at 37xc2x0 C. for 3-5 minutes. The mixed glial cell suspension was plated in either 75 or 162 cm2 culture flasks with DMEM containing 10% heat-inactivated FBS+120 U/ml P and 12 xcexcg/ml S and grown in a humidified 5% CO2 incubator at 37xc2x0 C. On day 7 of culture, flasks were shaken (90 rpm, 20 min, 37xc2x0 C.) and media exchanged. On day 14 and every 3-4 days thereafter, BM"PHgr" were detached using an orbital shaker (200 rpm, 2 hours, 37xc2x0 C., 5% CO2), then centrifuged (450xc3x97g, 25 min, 4xc2x0 C.). The pellet containing BM"PHgr" was resuspended gently with cold DMEM, cell number and viability assessed by trypan blue exclusion. Purified BM"PHgr" obtained by our method averaged greater than than 95% viability. Microglia were characterized as previously described (Mayer et al., 1999, supra).
Assay for BM"PHgr" O2xe2x88x92 generation. BM"PHgr" O2xe2x88x92 generation was determined by the superoxide dismutase(SOD)-inhibitable reduction of ferricytochrome C(FCC) (Mayer et al., 1999, supra). Briefly, spontaneous O2xe2x88x92 release from unstimulated BM"PHgr" was measured in the presence of FCC (50 xcexcM) and HBSS with or without SOD (700 units). To determine PMA-stimulated O2xe2x88x92 release, BM"PHgr" containing wells received FCC, HBSS and PMA [1 xcexcM] with or without SOD which inhibited greater than 95% of FCC reduction. All experimental treatments were run in triplicate and in a final volume of 1 ml. Changes in absorbance of BM"PHgr" supernatants were measured at 550 nm using a Beckman DU-650 Spectrophotometer. Differences in the amount of reduced FCC in the presence or absence of SOD were used to determine BM"PHgr" O2xe2x88x92 generation by employing the molecular extinction coefficient of 21.0xc3x97103 Mxe2x88x921 cmxe2x88x921.
LDH assay. To assess BM"PHgr" viability following incubation with vehicle (DMSO) or the manzamines, lactate dehydrogenase (LDH) release was determined spectrophotometrically as previously described (Mayer et al., 1999, supra).
Assay for BM"PHgr" thromboxane B2 (TXB2) generation. Following stimulation with PMA, TXB2 generation in the BM"PHgr" culture supernatants was measured using immunoassays (Cayman Chemical, Ann Arbor, Mich.) as indicated by the manufacturer""s protocol (Mayer, et al., 1999, supra).
Statistical analysis of the data. Data are expressed as meansxc2x1S.E.M. of triplicate determinations of the number of experiments indicated in the legend of each figure. Appropriate multiway analysis of variance was performed on all sets of data followed by Dunnett""s test.
Following are examples which illustrate procedures for practicing the invention. These examples should not be construed as limiting. All percentages are by weight and all solvent mixture proportions are by volume unless otherwise noted.